A method for readily isolating bacteria with cloned DNA is essential for successfully cloning DNA from any source. Identification of bacteria containing cloned DNA is most easily accomplished whenever the cloned DNA encodes a function that can be subjected to direct genetic selection, i.e., whenever survival of recombinant bacteria depends upon acquiring and expressing a function encoded by the DNA that is to be cloned. Identifying recombinant bacteria with cloned DNA is significantly more difficult when the cloned DNA does not encode a genetically selectable function because recombinant bacteria with cloned DNA must be identified against a potentially high background of recombinant bacteria that contain the cloning vector but lack DNA.
One approach to identifying recombinant bacteria with cloned inserts is to screen bacterial colonies for insertional inactivation of a reporter gene such as lacZ, the structural gene for .beta.-galactosidase (Sambrook et al., Molecular Cloning, A Laboratory Manual, p. 1.85-p. 1.86 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). .beta.-galactosidase can not be made when DNA is cloned into a restriction site that separates the coding portion of lacZ from its promoter. As a result, on medium with the chromogenic substrate X-gal, recombinants with cloned DNA appear as white colonies against a background of blue recombinant colonies that contain religated vector alone. Screening methods such as insertional inactivation of lacZ facilitate identification of bacteria with cloned DNA by flagging the appropriate colonies in some way. However, these methods do not favor or select for growth of bacteria with cloned DNA. Hence, whenever a screening procedure is used, it is usually necessary to look through large numbers of colonies to find a few with cloned DNA. A common strategy for reducing the number of background colonies is to prevent self-ligation of the vectors by using alkaline phosphatase to remove the terminal 5' phosphates from vector molecules because DNA ligase requires a terminal 5' phosphate to join the ends of DNA molecules (Sambrook et al. supra, p. 1.56-p. 1.58). This method generally reduces, but does not eliminate, background colonies lacking cloned DNA.
An approach that minimizes the number of bacteria lacking cloned inserts uses a vector that enables direct selection for recombinant bacteria with cloned DNA. The advantage of a selection method over a screening method is that growth of bacteria with cloned DNA is greatly favored over bacteria lacking cloned DNA.
Direct or positive selection vectors containing genes that convey lethality to the host are well known. For example, expression of the B. subtilis or the B. amyloliquefaciens sacB genes in the presence of sucrose is lethal to E. coli and a variety of other Gram-negative and Gram-positive bacteria [Cai et al., J. Bacteriol. 172, 3138, (1990); Gay et al., J. Bacteriol. 164, 918, (1985); Jager et al., FEMS Microbiol. Let. 126,1 (1995); Jager et al., J. Bacteriol. 174, 5462, (1992); Kamoun et al., Mol. Microbiol. 6,809, (1992); Kaniga et al., Gene 109,137 (1991); Ried et al., Gene 57,239, (1987); Simon et al., J. Bacteriol. 173,1502, (1991)]. The sacB gene encodes levansucrase [Gay et al., J. Bacteriol. 153,1424 (1983); Lepesant et al., Mol. Gen Genet. 128,213 (1974)]. Levansucrase catalyzes both the hydrolysis of sucrose and the polymerization of sucrose to form levan. The basis for the lethality of levansucrase in the presence of sucrose is not fully understood. However, the inability of E. coli and certain other bacteria to grow if sacB is expressed in the presence of sucrose means that inactivation of sacB can be used to directly select for bacteria that contain DNA inserted into sacB (Cai and Wolk, ibid.; Gay et al., supra.; Jager et al., supra.; Kaniga et al., supra.; Ried et al.,, supra; Simon et al., supra.]. Indeed, insertional inactivation of the wild type B. amyloliquefaciens sacB gene has been used to select for cloned DNA in E. coli [Pierce et al., Proceed. Natl. Acad. Sci. USA 89,2056 (1992)].
B. subtilis is an important alternative to E. coli for cloning of DNA. B. subtilis does not have lipopolysaccharide as a cell well component and, as a result, does not contaminate extracellular products with endotoxin [Harwood et al., p. 327-390. In C. R. Harwood and S. M. Cutting (eds.), Molecular Biological Methods for Bacillus (1990) John Wiley & Sons, New York]. Proteins secreted from B. subtilis are released into the culture medium rather than being trapped in a periplasmic space as is frequently the case for E. coli and other Gram negative bacteria (Harwood et al., supra). In addition to use as a tool for biotechnology, B. subtilis is intensively studied as a model system for cellular differentiation because of its ability to sporulate [(Errington, Microbiol. Reviews. 57,1 (1993)). Many sporulation genes have no counterparts in E. coli. As a result, the function of many sporulation genes can be properly examined only in B. subtilis.
Although the systems for genetic manipulation of B. subtilis are nearly as well developed as for E. coli, utilization of B. subtilis has lagged behind use of E. coli for cloning purposes. One problem is that identifying recombinant bacteria with cloned inserts is significantly more difficult with B. subtilis than with E. coli. Nicked plasmids, useful for genetic manipulations in E. coli, are inactive in transformation of B. subtilis [Bron, S., p. 75-174. In C. R. Harwood and S. M. Cutting (eds.), Molecular Biological Methods for Bacillus (1990) John Wiley & Sons, New York]. Consequently, B. subtilis cannot be transformed with vector molecules that have been dephosphorylated by alkaline phosphatase and ligated. This is a strategy commonly employed with E. coli for enrichment of molecules with inserts but cannot be applied to B. subtilis. Furthermore, the wild-type alleles of sacB from B. subtilis or B. amyloliquifaciens, useful in E. coli because of their lethality, cannot be used to select for cloned DNA in B. subtilis because expression of these alleles is not lethal for B. subtilis [Nagarajan et al., Res. Microbiol. 142,787 (1991)].
In an attempt to overcome the difficulty of genetic manipulations in Bacillus a variety of shuttle vectors have been developed for E. coli and B. subtilis so that DNA can be initially cloned in E. coli and then transferred to B. subtilis for subsequent manipulation (Bron, supra). However, it is well known that functional copies of certain genes can be expressed in B. subtilis but not in E. coli. In some cases, expression of a heterologous gene is toxic for E. coli. [Yudkin, Mol. Gen. Genet. 202,55 (1986); Ferrari et al., Proc. Natl. Acad. Sci. USA 82,2647 (1985); Hasnain et al., J. Gen Microbiol. 132,1863 (1986)]. Young et al., [p. 63-p. 103. In N. P. Minton and D. J. Clarke (eds.), Biotechnology Handbooks, Vol. 3, Clostridia (1989) Plenum Press, New York.]. Furthermore, there are a number instances where E. coli may be unsuitable for production of proteins encoded by certain heterologus genes. For example it would appear that B. subtilis is a preferred host for production of clostridial enzymes because overproduction of the Clostridium thermocellum celA gene product is known to result in rapid loss of viability for E. coli but not for B. subtilis [Schwartz et al., Appl. Microbiol. Biotechnol. 27,50, (1987); Soutschek-Bauer et al., Mol. Gen. Genet. 208:537-541 (1987)]. In other cases, differences in preferred codon usage may allow a gene to be translated in B. subtilis but not in E. coli [Garnier et al., Plasmid 19,134, (1988)].
There is a need, therefore, for vectors and methods that can be used to efficiently isolate recombinant molecules containing cloned DNA in B. subtilis without recourse to E. coli based systems. The problem to be solved is to develop a means of directly transforming Bacillus with plasmid DNA in such a way that transformants with inserts in the plasmid are selected for growth and transformants without inserts in the plasmid are non-viable. Applicant has solved this problem through the development of a positive selection vector for Bacillus. The selection vector makes use of a mutant levansucrase gene that confers lethality on the host Bacillus cell in the presence of sucrose. Insertion of foreign DNA into a cloning site within the mutant levansucrase gene results in the inactivation of the gene and allows the cell to grow. All cells transformed with vector molecules that lack foreign DNA inserts will not grow. In this fashion the vector permits the selection of colonies that contain inserts, while at the same time limiting the number of cells containing non-productive vector that must be screened.